Direct Complement-fixation Test Research Articles

Comparison of Procedures to Detect Chlamydia psittaci Antibodies in Cockatiels (Nymphicus hollandicus)

Diagnostic serologic procedures for psittacine chlamydiosis were evaluated using experimentally inoculated cockatiels (Nymphicus hollandicus). Chlamydia psittaci was recovered from subjects on day 16 postinoculation. Seroconversion was confirmed by the direct complement fixation (DCF) test. The blocking enzyme-linked immunosorbent assay was demonstrated to be the most sensitive of three serodiagnostic procedures evaluated from inoculation until day 24 postinfection, with 92% sensitivity and 73% specificity. The DCF test was progressively more sensitive over the duration of the experiment, attaining 100% sensitivity and specificity on the day 24 postinfection. This study confirms the ability of the DCF procedure to adequately detect C. psittaci antibodies. Limitations relating to reproducibility of serologic results under clinical conditions found in this study indicate that a further refinement of tests is required to consistently detect and quantify antibodies.

Bovine monoclonal antibody specific for Brucella abortus lipopolysaccharide

The development of a bovine monoclonal antibody against Brucella abortus smooth lipopolysaccharide (BM-8) by interspecies fusion of bovine peripheral lymphocytes from an immunized cow and a murine plasmacytoma cell line is described. The twice cloned cell line secreted bovine IgG 1 subclass antibody. Ascites fluid was prepared in pristane treated nu/nu mice by intraperitoneal injection. The pooled ascites fluid was purified by affinity chromatography and the functions of the antibody assessed in various serological tests. The BM-8 antibody did not agglutinate well at a neutral pH, however, under acid conditions it was efficient at agglutinating B. abortus cells. The antibody did not precipitate B. abortus LPS in double agar gel immunodiffusion but was very active in the direct complement fixation test and the indirect enzyme immunoassay, although it was unable to compete with a murine monoclonal antibody in a competitive enzyme immunoassay.

Antibody isotype response in adult cattle vaccinated with Brucella abortus S19

In a chronological study of sera collected from eight adult cattle vaccinated with 3 × 10 10 cfu of Brucella abortus S19, antibody of each of the four major isotypes was measured by indirect enzyme immunoassay (ELISA) and by direct and modified complement fixation tests (CFT). Six of the cattle gave antibody responses to the vaccine strain that commenced between days 5 and 8 for all the isotypes in the ELISA, peaked by 1 to 4 months and then declined to low levels by 10 months. Direct CFT and modified CFT titers were measurable by 7 or 8 days post-vaccination, and peaked by 1 month; direct CFT titers disappeared by 5 months while the modified CFT titers lingered for 10 months. Two animals gave cyclical direct CFT and modified CFT antibody responses, a cyclical IgG 1 response, a low IgG 2 and an elevated IgA response. The amplitude of the cycles was uniform over three cycles while the wavelength increased with time. A year post-vaccination, B. abortus S19 was isolated twice from milk from one of the animals (no attempt was made to culture B. abortus from the other). Sera from B. abortus naturally infected cattle were analysed for comparison.

The use of Elisa for detection of exogenous and endogenous avian leukosis viral antigens in basic breeding flocks

An enzyme-linked immunosorbent assay (ELISA) for avian leukosis/ sarcoma virus (ALV) group-specific (gs) antigens was used to study the identification of hens which congenitally excrete exogenous ALV. The sensitivity of this assay was compared with that of the phenotypic mixing test (PMT) and the direct complement fixation test (CFT) by testing limiting dilutions of purified avian myeloblastosis virus (AMV), embryo homogenates and albumens. About 0.4 ng/ml of purified AMV protein could be detected by ELISA and 23.8 ng/ml of AMV protein was demonstrable by the CFT. The lowest levels of gs-antigen detection corresponded with about 100 median tissue culture infectious doses (TCID50) of infectious ALV. Albumens and embryos of three White Leghorn flocks and one White Plymouth Rock flock were tested for the presence of avian leukosis virus (ALV) gs-antigens by the complement fixation test (CFT) and the enzyme-linked immunosorbent assay (ELISA) and exogenous ALV employing the phenotypic mixing test (PMT). The highest number of ALV-gs antigen positive samples of egg albumens was obtained by the ELISA. In embryo homogenates prepared from eggs of the four flocks under study 100% scores for gs-antigens were obtained by ELISA. A differentiation between gs-antigens of endogenous and exogenous ALV was made by testing of supernatant fluids after one chick embryo fibroblast passage of the C/E phenotype. Endogenous viral (ev) genes leading to the expression of endogenous ALV gs-antigens were apparently present in practically all chickens of the four flocks under study. Complete endogenous ALV (subgroup E) was detected in embryos (41%) from the White Plymouth Rock grandparent flock, but was not found in embryos of two White Leghorn basic breeder flocks. The results indicate that both flocks of White Leghorn chickens are endowed with ev 3 genes. Circumstantial evidence was obtained for the prevalence of endogenous ALV gs-antigens in albumen samples of eggs from flocks with gs+chf+ and V-E+ phenotype respectively.

An Enzyme-Linked Immunosorbent Assay for Detecting Avian Leukosis-Sarcoma Viruses

Immunoglobulins from antiserum raised against chromatographically purified avian myeloblastosis virus (AMV) group-specific (gs) antigens were used in enzyme-linked immunosorbent assay (ELISA). Readily discernible color was produced with 2--3 ng of AMV protein in microplate wells coated with 4 micrograms of salt-precipitated immunoglobulins. When a biological assay, i.e., phenotypic mixing (PM), was the criterion for the infectious status of specimens, the ELISA consistently identified a greater percentage of virus-positive specimens than direct complement-fixation (DCF) tests. Over 95% concordance was obtained between the ELISA and PM bioassays when meconia and whole-blood samples were tested. Moreover, three DCF(-) egg albumens from one virus shedder hen were positive by the direct ELISA. Complete agreement was found between a biological assay for endogenous virus and the ELISA when blood and albumens from inbred chickens were tested. The ELISA is a rapid and convenient alternative to the DCF test for identifying infected chickens in eradication programs, because virus-rich sources such as meconia and blood that are unsuitable for DCF can be tested directly.

Studies of Flocks with High Mortality from Lymphoid Leukosis

Two pairs of commercial white-egg parent flocks were selected for study, because one of each pair was observed to be dying with lymphoid leukosis at a high rate. The proportion of each flock producing eggs with lymphoid leukosis virus in the albumen was studied. In one pair the rate of birds shedding was no different in the high- and low-mortality flocks. The total rate of shedding was lower in the other pair, but the rate was higher in the high-mortality flock. These data indicate that the rate of virus infection is not always proportional to the development of lymphoid leukosis, and that other environmental factors may play a role. Direct complement-fixation tests on the albumens which were positive for lymphoid leukosis virus showed that group-specific antigen could be detected in 83%. Therefore, the direct complement-fixation test can be used on albumens as a rough estimate of shedding rate.

A Comparative Examination of Swine Sera for Antibody To Aujeszky Virus with the Conventional and a Modified Virus-Serum Neutralization Test and a Modified Direct Complement Fixation Test

Sera of pigs from élite breeding herds, of boars and sows collected at slaughter-houses, and of pigs from herds known to be infected, were examined for antibody to Aujeszky virus. The conventional and a modified virus-neutralizing antibody (VNA) test and a modified direct complement fixation (CF) test were employed. In simultaneous titrations of positive sera the modified VNA test gave titers approx. 4 log2 units above the titers obtained by the conventional test. The conventional VNA test was found insufficiently sensitive. Unspecific neutralization in the modified VNA test was infrequent in serum dilution 1/2 and rare in dilution 1/4. The GF tests on sera of slaughter sows and animals from known infected herds showed a remarkable consistency with the VNA tests. Inconsistent results were obtained with but few sera. Abt. 5 % of the sera could not be examined because of complement fixation with control antigen.

A direct complement fixation test for infectious bronchitis virus using heat-inactivated chicken sera.

A direct complement fixation test for infectious bronchitis virus using heat-inactivated chicken sera.

Avian Complement-Fixation

The complement-fixation (CF) test has been used extensively for studies of diseases in man and animals. It is used to detect antibodies or antigens resulting from natural or experimental infections. A large number of samples may be tested, giving rapid, dependable results if appropriate controls are used. Complement-fixation tests have been used less extensively with avian systems, partly because of technical difficulties encountered with the antisera. Unheated antisera are frequently anticomplementary (AC), whereas heated avian antisera usually do not fix complement (C') in the conventional direct manner. Therefore, indirect and modified direct tests have been devised. Each test has its advantages and disadvantages. Some theoretical aspects of the CF technique are covered, along with practical application of the test using avian sera. The modified direct CF test is discussed in some detail. The CF test should be utilized in research and diagnostic laboratories concerned with problems of avian disease.

A Modified Direct Complement-Fixation Test for Detection of Marek's Disease Antibodies in Chicken Serum

A modified direct complement-fixation (CF) test is described which will detect antibodies in chicken serum associated with Marek's disease herpesvirus (MDHV) infection. The test is modified in that normal chicken serum (NCS) is incorporated into guinea pig complement (C') in an optimal quantity, as determined by titration, to obtain a maximal antiserum or antigen titer. Suitable CF and agar gel precipitin (AGP) antigens were prepared from saline infusion preparations of feather tips from chickens infected with MDHV. Specific and stable antigens in sufficient quantities were obtained by this method over a period of 3 to 18 weeks. Comparative CF and AGP tests on sera from chickens infected with MDHV, contact chickens, and naturally exposed chickens revealed that AGP antibodies were detectable earlier than CF antibodies. The correlation between the 2 tests improved with time after MDHV exposure. The results suggest that the type of antibodies detected by the 2 tests differ qualitatively. Both tests appeared to be specific for detecting MD infection.

A Direct Complement-Fixation Test for Detection of Mycoplasma Antibodies in Chicken Serum

SUMMARY A direct complement-fixation (CF) test is described that utilizes Mycoplasma gallisepticum (MG) antigen and chicken antiserum. Fixation of guinea pig complement (C') was achieved by incorporating untreated normal chicken serum (UNCHS) directly into guinea pig C'. The factor(s) in normal chicken serum which is responsible for CF could be titrated to obtain maximal serum titers. This factor(s) appears to be relatively labile to freezing and/or thawing, but stable at refrigerator temperature for at least 6 weeks. The CF test was compared with the MG serum-plate (SP) and hemagglutination-inhibition (HI) tests on sera obtained from chickens infected with MG and Mycoplasma synoviae (MS) isolates as well as sera obtained from field flocks. Correlation was good among the three tests. Little cross-reactivity of MG and MS antibodies by CF was encountered. The sensitivity and specificity of the CF test equalled or surpassed the HI test. Preliminary investigations indicate that the CF test may find application in detecting antibodies in chicken sera to some common avian viral agents.

Direct complement fixation test with avian infectious bronchitis virus in chicken.

The direct complement fixation test was performed to follow the antibody response in chickens infected with avian infectious bronchitis virus. Concentrated allantoic fluid (4 units) was used as an antigen and allowed to react with serially diluted antiserum in the presence of two complete units of guinea-pig complement for 3 hr. at 4 degrees C. and (1/2) hr. at 37 degrees C. before the addition of sensitized cells. Serum was unheated and used either fresh or within one month of storage at -30 degrees C. Individual birds showed a rise and fall of complement-fixing antibody both after primary and secondary inoculations. The complement-fixing antibody was detected as early as the seventh day after primary inoculation. The highest complement fixation titre (1/32 to 1/64) was recorded from 14 to 21 days after inoculation with a subsequent gradual decline.The results of the direct complement fixation tests have been correlated with the serum neutralization test. The neutralizing antibodies usually appeared by the 14th day but were not detected at a significant titre until the 21st day after primary inoculation. Serum neutralizing antibodies were still present at high titres even after 7 weeks of infection but the complement-fixing antibodies had disappeared by that time.

Antibody Response of Adult Chickens to Infectious and Noninfectious Foot-and-Mouth Disease Virus

Summary Lesions suggestive of infection were observed in or on the mouth, combs and feet of chickens inoculated with Type A-119 virus propagated in tissue culture. Lesions were not observed in chickens inoculated with Type A-GB virus propagated in guinea pig footpads. A high titer of neutralizing antibody was induced in adult chickens by inoculation of 1 and 2 ml of either infectious or noninfectious Type A-119 virus grown in tissue culture. Infectious Type A-GB, propagated in guinea pig pads, produced a relatively low titer of neutralizing antibody in chickens. Both strains, 119 and GB, of infectious Type A virus produced a good response of complement-fixing antibody in chickens as tested by a modified direct complement fixation test. No evidence of infection was found in normal chickens held with infected birds in the same cage.

A Modified Direct Complement Fixation Test for the Detection of Antibodies in the Serum of Cattle Previously Infected with Vesicular Stomatitis Virus

Summary A modified direct complement fixation method for the detection of antibodies in heat-inactivated sera of cattle infected with vesicular stomatitis virus is described. The addition of fresh unheated bovine serum to the diluted guinea pig complement constitutes the only modification. In its absence no fixation of complement is observed. The normal serum supplying the apparently essential heat-labile factor, is pretested for absence of nonspecific reactivity with test antigen. Sera of animals around 6 months of age show less nonspecific reactivity than sera of adult cattle. This method when applied in testing serial bleedings from cattle infected with either the New Jersey or Indiana type of vesicular stomatitis virus, detected the antibodies 9 to 10 days after intralingual infection. Antibodies were demonstrable at a diagnostic level for at least 90 days afterward. The test was shown to be sufficiently specific to indicate the type of virus in epithelial tongue tissue collected at the acute stage of infection and to reveal the type-specificity of the antibodies in serum of convalescent cattle. In indirect complement fixation tests, the inhibition titers of early convalescent sera were much lower than those recorded in the modified direct complement fixation test. The difference was negligible with hyperimmune sera.

A Capillary Tube Agglutination Test for Detecting Antibodies against Ornithosis in Turkey Serum

Summary and Conclusions A capillary tube agglutination test using a Giemsa-stained, buffered suspension of elementary bodies prepared from a toxigenic turkey strain of ornithosis virus propagated in the allantoic cavity of chicken embryos was compared with the indirect complement fixation test, and the results were promising. The CTA test seemed to be more sensitive in the first week or two after infection, and thereafter the two tests seemed equally sensitive. Both were a little more sensitive than the direct complement fixation test when the test material was turkey serum. In the CTA test false positive reactions were encountered, usually in low serum dilutions, but occasionally in dilutions as high as 1:32, when the serum was hemolyzed, contaminated or contained residual fibrin. Reactions at dilutions of 1:16 or higher constituted good presumptive evidence of infection or of some previous encounter with ornithosis agents; reactions at dilutions of less than 1:16 were less specific and were interpreted cautiously. Preliminary testing of serum from infected parrots and pigeons indicated that the CTA test can detect ornithosis antibodies in these species. The CTA test is economical and simple to perform. According to the results of these tests it could be a useful flock test, of value in epizootiologic surveys, and a rapid screening test of flocks before marketing in enzootic or epizootic areas. Flocks undergoing acute or subacute epizootics of ornithosis, the most dangerous from the standpoint of public health, could be detected easily.

Direct complement-fixation test for diagnosis of ornithosis in turkeys.

SummaryEmploying a soluble antigen, complement-fixation reactions were obtained with turkey antiornithosis sera. Antigens consisting of viable allantoic and killed yolk-sac virus suspensions did not show the same degree of activity. The direct complement-fixation reaction was as sensitive as the indirect complement-fixation test; therefore, the use of the direct test for the study and diagnosis of turkey ornithosis was suggested.

Studies of the Complement Fixation Reaction in Virus Systems

Summary Sera collected from horses, swine, and cattle before inoculation with either the New Jersey or Indiana types of vesicular stomatitis virus and at periodic intervals thereafter, were tested by complement-fixation methods with antigens prepared from chick embryo tissues infected with these respective viruses. Convalescent sera from the three species differed in their behaviour in these tests. In horses as in guinea pigs, complement-fixing activity with homologous VS antigen appeared in about six to eight days after infection, reached a maximum titer in ten days to two weeks, remained relatively constant for seven to ten days then began to decline. No cross fixation between types was recorded. None of the cattle sera at any time exhibited any fixation in direct complement-fixation tests with homologous antigen. To demonstrate non-complement-fixing antibody in these convalescent bovine sera it was necessary to employ an indirect complement-fixation method using hyperimmune guinea pig or convalescent horse serum to detect residual antigen. The time of antibody development as indicated by the appearance of inhibitory activity in these indirect tests coincided with that demonstrated in horses by direct complement-fixing tests. The convalescent pig sera showed marked prozones effects (increased hemolysis) in lower serum dilutions. This increased hemolysis was traceable to certain substances present in normal as well as in convalescent swine serum and not to non-complement-fixing antibodies corresponding to those in convalescent bovine sera. Natural hemolysins for sheep red cells, third component of complement, and possibly other constituents, notably lipids, apparently contribute to this augmented hemolysis or reduced fixation of complement.

Studies of the Complement-Fixation Reaction in Virus Systems

Summary In direct complement-fixation tests, the quantitative behaviour of FMD viral antigens with hyperimmune guinea-pig sera was found to be similar to that noted in a number of viral antigen-antibody systems previously studied. The relative complement-fixing capacity of several FMD antigens with different guinea-pig antisera was estimated from the slope of the complement-antigen regression lines. Such a comparison showed a much closer resemblance in the complement-fixing behaviour of the strain of A type FMD virus isolated during the recent Canadian outbreak and an A type strain isolated in England at about the same time, than either displayed toward a stock A strain, No. 119, that had been maintained for many years by cattle passage. Sera of only two of the convalescent cattle tested, exhibited fixation of guinea-pig complement with FMD virus antigen. The relatively rapid development of inhibitory or neutralizing activity in the sera of cattle infected with FMD virus could be detected, however, by an indirect complement-fixation method with FMD antigens; pre-infection bleedings from the same animals did not show this activity. In lower dilutions, these convalescent sera blocked or neutralized the complement-fixing activities of FMD viral antigens of heterologous as well as of the homologous type. In higher serum dilutions the inhibitory activity extended only to virus of the homologous type. At the present time, there is an evident need for a satisfactory in vitro method of demonstrating antibody in the sera of cattle convalescent from foot-and-mouth disease. The direct complement-fixation test has proved of limited value for this purpose since only a small proportion of cattle develop complement-fixing activity with FMD antigens. The preliminary studies presented here suggest that the indirect complement-fixation test, particularly if used in parallel with one of the direct form, merits further investigation from this practical standpoint.

補体結合阻止反応に関する研究

As the results of a prelininary test it was found that the specificity in the direct complement fixation test run on pigeon sera obtained by immunization with the pigeon adapted newcastle disease virus (infected pigeon brain) was observed by the complement dilution method described already in our previous paper.In this experiment the antigen was prepared from infected embryo and chorioallantoic fluid purified by simple centrifugation (4000 r.p.m. 20 minutes). Then using the pigeon sera and the antigen, specific C-F-I-T was carried out on chicken sera obtained by the vaccination with the living attenuated virus.As regard to the results of these tests it was found that the complement fixation inhibitory antibodies were not always paralleled to that of hemagglutination inhibitory antibodies, and that the former was demonstrated generally earlier than the latter.The fowl plague virus were distinguished clearly from the newcastle disease virus by means of the C-F-T, using the method of the direct complement dilution.

補体結合阻止反応に関する研究

As the results of a prelininary test it was found that the specificity in the direct complement fixation test run on pigeon sera obtained by immunization with the pigeon adapted newcastle disease virus (infected pigeon brain) was observed by the complement dilution method described already in our previous paper.In this experiment the antigen was prepared from infected embryo and chorioallantoic fluid purified by simple centrifugation (4000 r.p.m. 20 minutes). Then using the pigeon sera and the antigen, specific C-F-I-T was carried out on chicken sera obtained by the vaccination with the living attenuated virus.As regard to the results of these tests it was found that the complement fixation inhibitory antibodies were not always paralleled to that of hemagglutination inhibitory antibodies, and that the former was demonstrated generally earlier than the latter.The fowl plague virus were distinguished clearly from the newcastle disease virus by means of the C-F-T, using the method of the direct complement dilution.